Sustained calcium entry, in many cells, is fundamental to the initiation and maintenance of specific cellular responses. In addition to the widespread store-operated calcium influx mechanism, whose molecular nature remains elusive, other putative calcium influx pathways have emerged through identification of a growing number of genes coding for calcium-permeable cation channels. Our preliminary data describe the molecular characterization of a novel calcium-entry pathway controlled by ADP-ribose (ADPR). This characterization includes the identification of a novel highly specific ADP-ribose hydrolase, NUDT9, which shares high homology with the C-terminal region of a previously identified gene, LTRPC2, and the functional demonstration that LTRPC2 encodes a protein product that is an ADPR-gated calcium-permeable cation channel. We also identified natively expressed ADPR-dependent conductances in pancreatic beta cells and human monocytes. Based on these data, we propose to explore the mechanisms that regulate ADPRmediated calcium entry in recombinant and physiological systems. In Specific Aim 1, we will analyze the biophysical and molecular aspects of LTRPC2 ion channel function by using a combination of calcium imaging and electrophysiological analysis of cells that express recombinant LTRPC2. To identify the structural basis for ADPR-dependent gating of LTRPC2, we will express and characterize truncated or chimeric LTRPC2 constructs directed towards the C-terminal nudix domain, the putative ADPR binding region. We also propose to systematically alter amino acids within this domain to assess structure-function relationships that confer selectivity for ADPR gating. In Specific Aim 2, we will address the functional and physiological role of ADPR-gated channels in the regulation of calcium homeostasis of beta cells. We will investigate the specific properties of native ADPR-gated channels and compare them with those of recombinant LTRPC2. We will assess their functional role in the cellular responses of the above cells by comparing the relative contributions of ADPR-gated Ca2+ signals to those of store-operated Ca2+ influx and voltage-dependent Ca2+ channels. Finally, we will seek to identify the mechanisms responsible for ADPR production by investigating the major enzymes and pathways involved in its metabolism.